Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation
- Pfizer Inc, United States
- University of Oxford, United Kingdom
Figures
Figure 1 with 5 supplements
Cryo-EM structure of human SLC1A5.
(a) A density map of SLC1A5 homotrimer viewed from the side of the membrane (left) and the extracellular face (right) highlighting the scaffold domain (green), transport domain (navy), ECL2 (red) …
Figure 1—figure supplement 1
SLC1A5 protein purification, functional assays in cells and proteoliposomes and negative staining.
(a) Uptake of [14C]-glutamine in HAP1 SLC1A5KO cells or SLC1A5KO cells transiently transfected with pcDNA3 vector containing the indicated sequence in the presence (solid bar) or absence (open bar) …
Figure 1—figure supplement 2
Cryo-EM analysis of SLC1A5-cKM4012 (Fab) complex.
(a) Flow chart of the cryo-EM data processing procedure. Details can be found in the Materials and methods. (b) A representative cryo-EM micrograph. (c) Local resolution of the map estimated using …
Figure 1—figure supplement 3
Cryo-EM analysis of SLC1A5-cKM4012 (Fab) complex in the presence of L-glutamine.
(a) Flow chart of the cryo-EM data processing procedure. Details can be found in the Materials and methods. (b) A representative cryo-EM micrograph. (c) Local resolution of the map estimated using …
Figure 1—figure supplement 4
Cryo-EM densities of the eight transmembrane helices with ECL loops of SLC1A5-cKM4012 (Fab).
(a) Cryo-EM density is sharpened with a negative b-factor 15 Å2 and displayed at the contour level 8σ for the transmembrane helixes and loops regions, except for the ECL2a, and b regions at 5.6σ and …
Figure 1—figure supplement 5
Sequence alignment of human SLC1 transporters and two prokaryotic homologues.
The secondary structural features are presented according to the human SLC1A5 cryo-EM structures in this study. The color-coding scheme is the same as in Figure 1d. The secondary element changes on …
Figure 2
Structural comparison between outward- and inward-facing states of SLC1A5.
(a and b) Structure of the SLC1A5 monomer viewed from the side of the membrane highlighting TM6 and TM3, respectively. Transport domains are represented as molecular surfaces and colored in navy. …
Figure 3 with 4 supplements
The substrate, L-glutamine-bound SLC1A5 structure.
(a) Conformational change for the HP2 upon binding of L-glutamine in the outward-facing state of SLC1A5. The unliganded structure is shown in gray and the L-glutamine-bound structure is in navy. EM …
Figure 3—figure supplement 1
The extracted ion chromatograph (above) and the mass spectrum (below) for CHS is shown in negative ion mode.
CHS is measured with a three ppm accuracy in the SLC1A5 sample. The exact mass, elution time, and fragmentation pattern are validated against a commercially bought standard (Sigma-Aldrich, St. Louis).
Figure 3—figure supplement 2
Potential CHS interaction in outward- and inward-facing conformation of SLC1A5.
(a) Elongated densities, shown as blue mesh (~6σ), are observed between two protomers of SLC1A5 trimer in both the outward- and inward-facing (PDB: 6GCT) cryo-EM structures. (b) A density is …
Figure 3—figure supplement 3
Cryo-EM densities of the substrate and putative CHS binding sites of SLC1A5, at the contour level 6σ.
(a) Cryo-EM densities of the substrate-binding site with (upper panel) or without (lower panel) Gln substrate. (b) Cryo-EM densities of the putative CHS binding site with (upper panel) or without …
Figure 3—figure supplement 4
Selective examples of allosteric druggable pockets at the lipid-exposed surface near the intracellular part of membrane proteins.
Different protomers are shown in surface with different gray scale. Each ligand is colored in yellow ball-and-sticks. Examples of allosteric sites include the RTX agonist binding site in the …
Figure 4
Schematic representation of conformational changes in SLC1A5 during substrate transport.
(a) In the outward-facing state (top), the Zone two from the transport domain interacts with the scaffold domain, with the Zone one partially occupied by the ECL2a. Two different poses of the ECL2a …
Tables
Table 1
Data collection, reconstruction, and model refinement statistics
https://doi.org/10.7554/eLife.48120.002| SLC1A5_cKM4012 EMD-9187, PDB: 6MP6 | SLC1A5_cKM4012_L_Gln EMD-9188, PDB: 6MPB | |
|---|---|---|
| Data collection | ||
| Microscope | Titan Krios | Titan Krios |
| Voltage (keV) | 300 | 300 |
| Nominal magnification | 22,500 x | 22,500 x |
| Exposure navigation | Stage Position | Stage Position |
| Electron exposure (e /Å2) | 42 | 42 |
| Dose rate (e/pixel/sec) | 5 | 5 |
| Detector | K2 Summit | K2 Summit |
| Pixel size (Å)* | 0.543 | 0.543 |
| Defocus range (µm) | 1.2 to 2.5 | 1.2 to 2.5 |
| Micrographs Used | 3804 | 5228 |
| Final Refined particles (no.) | 165,067 | 253,220 |
| Reconstruction | ||
| Symmetry imposed | C1 | C1 |
| Resolution (global) | ||
| FSC 0.143 | 3.50 Å | 3.83 Å |
| Applied B-factor (Å2) | −15 | −15 |
| Refinement | ||
| Protein residues | 1341 (SLC1A5) | 1344 (SLC1A5_L_Gln) |
| Map Correlation Coefficient | 0.815 | 0.785 |
| R.m.s deviations | ||
| Bond lengths (Å) | 0.006 | 0.005 |
| Bond angles (°) | 1.085 | 1.062 |
| Ramachandran | ||
| Outliers | 0.00% | 0.00% |
| Allowed | 7.88% | 8.93% |
| Favored | 92.12% | 91.07% |
| Poor rotamers (%) | 0.00% | 0.00% |
| MolProbity score | 1.62 | 1.67 |
| EMRinger score | 2.59 | 1.99 |
| Clashscore (all atoms) | 3.44 | 3.67 |
-
*Calibrated pixel size at the detector
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Mouse monoclonal Anti-SLC1A5 | Creative Biolabs Inc | TAB-1010CLV | 1 ml, 1 mg/ml |
| Strain, strain background (Homo sapiens) | HEK293S GnTI- cells | ATCC | CRL-3022 RRID: CVCL_A785 | |
| Recombinant DNA reagent | pcDNA3.1+ | Life Technologies | V79020 | |
| Chemical compound, drug | Opti-MEM reduced serum media | Life Technologies | 31985062 | |
| Strain, strain background (Homo sapiens) | HAP1 SLC1A5 knock out cells | Horizon Discovery | HZGHC005452c002 | |
| Chemical compound, drug | Iscove’s modified Dulbecco’s medium | Gibco | 12440 | |
| Chemical compound, drug | Cell dissociation buffer | Gibco | 13151 | |
| Chemical compound, drug | Fetal bovine serum | Gibco | 16000044 | |
| Software, algorithm | MotionCorr2 1.1.0 | Zheng et al., 2017 | http://msg.ucsf.edu/em/software/motioncor2.html | |
| Software, algorithm | Relion v 2.0 | Kimanius et al., 2016 | https://www2.mrc-lmb.cam.ac.uk/relion/ | |
| Software, algorithm | Phenix 1.14 | Adams et al., 2010 | http://phenix-online.org/ | |
| Software, algorithm | Coot 0.8.9.1 | Emsley and Cowtan, 2004 | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | |
| Software, algorithm | Pymol 2.0 | Schrodinger LLC | https://pymol.org/2/ | |
| Software, algorithm | Chimera 1.12 | Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera/ | |
| Software, algorithm | Gctf | Zhang, 2016 | https://www.mrc-lmb.cam.ac.uk/kzhang/ | |
| Software, algorithm | cisTem | Grant et al., 2018 | https://cistem.org/ |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.48120.016
